The rapid development of molecularly targeted probes for use in vivo has led to growing interest in clinical applications that incorporate information from these probes. In particular, there is a need for techniques to visualize these targeted probes during surgery, particularly to identify tissue either for removal or preservation. When looking at fluorescent probes in tissue, the major difficulty is usually separating the probe fluorescence from the tissue autofluorescence. Because the autofluorescence has different spectral properties than the fluorophore, it can easily be separated using multispectral imaging, in which several full emission spectra can be taken for each pixel. Although the implementation of acousto-optic tunable filters has greatly increased the speed of these techniques, the minimum time required to acquire an image cube is still several seconds with commercially available systems. When imaging using fluorescent probes with lower efficiency or when used at lower doses, the image acquisition time can be as long as several minutes. This data acquisition rate makes the use of multispectral imaging to provide real-time feedback during a surgical procedure impractical. Even when used for diagnostic purposes, the unavoidable motion of the subject during the time required to acquire an image cube can lead to an unacceptable loss of spatial resolution. This project focuses on the development of instrumentation for incorporating fluorescent and multi-spectral imaging into surgical applications, using two major approaches. The first approach is to develop a system for real-time visualization of fluorescent probes to guide surgery. The second approach is to develop a system for diagnostic applications, to provide a white-light stack of images for spatial registration of the multispectral image cube. As an alternative to multispectral imaging, aggressive filtering of both emission and excitation light can help to minimize the effects of tissue autofluorescence. Although the signal level drops as the spectral window is narrowed, this can be overcome by using a sensitive camera, such as a cooled CCD, ICCD, or even an EMCCD. Because a single spectral window is used, substantially faster data acquisition is possible. The immediate application for this instrument was the identification of peritoneal metastases in ovarian cancer, using a GSA-Rhodamine Green probe developed in NCI. The first prototype instrument, used for mock cytoreductive surgery in a murine model of ovarian cancer, had sufficient sensitivity to identify labeled metastases at an image acquisition rate of five frames per second with specificity comparable to that achieved with a multispectral system. An improved prototype, which permitted image acquisition at fifteen frames per second with enhanced sensitivity, was used for cytoreductive surgery on anesthetized animals with similar success to the previous mock surgery. This year, we also continued development on a separate multispectral system to provide a white light image stack for physical registration of the multispectral image cube, using a 92:8 beam splitter and a low-cost monochrome CCD camera in parallel with the multispectral instrumenation. Preliminary experiments on a cervical cancer animal model are underway. The hardware developed for this application could be easily adapted to incorporate a second camera into the instrument for fluorescence guided surgery;this second camera could be used either for a second fluorescence image, in order to provide a simple autofluorescence correction, or for a pseudo-white light image of the surgical field. Finally, some initial proof-of-principle in vivo imaging experiments were performed using upconverters, in this case upconverting nanocrystals that absorb 980 nm light and emit in the visible to near-infrared. These novel compounds could allow single-wavelength imaging with no detectable autofluorescence;the tissue background is so low that a white light image will likely be required to provide anatomical reference. Although considerable work remains in developing these nanoparticles for medical applications, this is a promising avenue for future efforts.